Variations in monoamine oxidase (MAO) activities in humans have been implicated as having a role in a number of human diseases, including alcoholism, psychiatric illness and Parkinson disease. Levels of activity for both forms of the enzyme, MAO-A and MAO-B, are genetically determined, as measured in skin fibroblasts and platelets, respectively; and both forms are critical to the metabolism of biogenic amines in the central nervous system and throughout the body. Full length cDNA sequences for MAO-A and MAO-B have been described and both genes have been mapped to the p11 region of the human X chromosome. The proposed studies will focus on the development of new molecular genetic techniques to resolve whether allelic variations exist for nucleotide and deduced amino acid sequences of MAO-A and MAO-B. New techniques will involve polymerase chain reaction (PCR) amplification of cDNAs combined with direct sequencing, chemical cleavage and denaturing gel electrophoresis to detect single base pair differences. Synthetic oligonucleotide primers will be used for first-strand cDNA synthesis of overlapping regions of 200-500 bp MAO-A and MAO-B mRNAs from skin fibroblasts and lymphocytes, respectively followed by PCR amplification. GC-rich clamps will be joined to the primers to increase the sensitivity of denaturing gels to detect differences among homo- and heteroduplex cDNAs from different individuals. Chemical cleavage will be carried out on heteroduplex cDNAs from pairs of male samples using a Maxam- Gilbert sequencing ladder to pinpoint the region of variation. Direct sequencing will be carried out by the dideoxy method using synthetic primers. These studies will provide a basis to establish whether allelic variations in the MAO loci exist and contribute to hereditary variations in MAO activity levels in the human population. The identification of sequence differences will also allow assessment of the correlation between specific MAO alleles and human disease states in larger populations, whether these differences occur in coding or regulatory elements of the MAO genes.